This program project is designed to determine the interaction among several factors which are involved in the regulation of the release of neurotransmitters. Experiments are described using resealed synaptosomes as representative of nerve endings to define the effect of various regulatory factors on release of neurotransmitters. Subcellular systems which regulate calcium transport will be studied, since calcium is an absolute requirement for exocytotic release. The electrogenic properties of Na ion-Ca ions exchange will also be measured. A number of regulatory systems are known to be involved in affecting neurotransmitter release. These include factors such as adenosine, norepinephrine, acetylcholine, and prostaglandins. The interactions of these factors will be observed at a number of organizational levels from the physical properties of nerve ending membranes to nerve stimulated release in perfused organs. The role of membrane lipids in the release of neurotransmitters will be investigated by feeding diets with varied lipid composition to pregnant rats and thereby altering lipid composition of membranes in the developing pups. Changes in membrane lipids will also be correlated with changes in calcium flux which occurs during exocytosis. It has recently been suggested that methylation of proteins may be involved in exocytosis. Methylation of phospholipids is known to affect membrane fluidity in erythrocytes. We have recently found that phospholipid methylation can also occur in brain and experiments are designed to determine whether presynaptic regulation of exocytosis could be mediated by phospholipid methylation. The final portion of this project is to develop new and more sensitive methods for measuring exocytotic release of neurotransmitters. Experiments are designed to adapt state-of-the-art electrochemical methods and flow injection analysis for the measurement of neurotransmitter release. It is hoped that more precise sequencing of the events which occur in exocytosis can be determined through the use of these techniques.